The goal of this revised research project is to understand the mechanisms underlying cell-to-cell HIV-1 infection through T cell virological synapses (VS). T cell VS are adhesive structures formed between infected and uninfected cells that are initiated by the molecular engagement of Env and CD4. Transmission through T cell virological synapses is a predominant mode of viral spread in cell culture and likely is equally important in vivo where T cell density and motility are more pronounced. Efficient cell-to-cell transmission through VS requires active cellular processes that coordinate viral assembly with transfer of nascent virions into recipient cells. While the same viral proteins mediate infection b cell-free virus and through the VS, the VS transfers more viral material directly from cell to cell and does so while evading many antibody responses. We suggest that signaling through the Env cytoplasmic tail (CT) coordinates cell-cell infection so that Gag, Env and genomic RNAs are assembled and transferred together as an infectious virus. Using novel Env imaging techniques in conjunction with reagents to image RNA genome packaging, we propose mechanistic studies to test how viral assembly and transfer of virus across the VS are coordinated. We have learned that infection through the VS can resist neutralizing antibodies and will examine the structural determinants that may support such resistance. We will test an inside out allosteric model whereby the ability of the Env CT regulate fusion also allows it to resist antibodies. Through systematic mutagenesis of the Env CT, we have identified mutants with a selective loss of the capacity to initiate infection via cell-free route while preserving its ability to infect via the cll-to-cell route and vice versa. These mutants will be used to directly probe for key interactions between Env and Gag that regulate cell-cell transmission. In addition these selective mutants will allow us to dissect the relative contributions of cell-to-cell versus cell-free transmission fr efficient HIV spread within primary human lymphoid tissue. The overarching hypothesis of this grant is that the Env CT coordinates HIV-1 assembly and budding in T cells to enhance the transfer of infectious virus and to minimize exposure to sensitive viral epitopes during cell-cell infection. Because the VS may underlie efficient in vivo transmission that resists humoral immunity, identifying the most sensitive targets may be critical for developing more effective vaccines and drugs.